DNA molecules and vectors regarding clavulanic acid biosynthesis enzymes

ABSTRACT

DNA sequences obtained from S. clavuligerus, recombinant vectors incorporating such sequences and hosts transformed with such vectors are disclosed. The DNA comprises one or more genes encoding one or more enzymes involved in the biosynthesis of clavulanic acid and may be used to improve the yield of clavulanic acid produced by clavulanic acid-producing organisms such as S. clavuligerus ATCC 27064.

This application is a continuation of application Ser. No. 921,352,filed Jul. 24th, 1992, now abandoned, which is a continuation ofapplication Ser. No. 664,460, filed Mar. 1st, 1991, now abandoned, whichis a continuation of application Ser. No. 358,671, filed, May 30th,1989, now abandoned, the disclosures of which are all incorporatedherein by reference.

The present invention relates to DNA molecules, especially recombinantDNA molecules, and in particular to recombinant vectors for use in thetransformation of a microbial host which contain inserted DNA fragmentscarrying one or more genes coding for enzymes involved in clavulanicacid biosynthesis.

Clavulanic acid is a potent β-lactamase inhibitor produced by themycelial bacterium Streptomyces clavuligerus (see Reading, C., and Cole,M., Antimicrobial Agents and Chemotherapy, 1977, 11, 852-857) and is acompound of great clinical value, since it protects β-lactamase-labileβ-lactam antibiotics from degradation. Methods for increasing the yield(titre) of clavulanic acid in fermentation processes are potentially,therefore, of considerable commercial importance.

One approach to the problem of clavulanic acid yield improvement thathas been envisaged involves the use of recombinant DNA techniques usingS.clavuligerus as the host cell. In this connection, it was suggestedthat isolation of genes involved in clavulanic acid biosynthesis fromthe chromosomal DNA of S. clavuligerus would be a possible startingpoint (see C. R. Bailey et al, Biotechnology, 1984, 2, 808-811).Hitherto, however, no DNA has been specifically identified as being ofvalue for increasing clavulanic acid titre.

Although several enzymes are believed to be involved in clavulanic acidbiosynthesis, only the enzyme clavaminic acid synthase (or synthetase)has so far been characterised. Clavaminic acid synthase (hereinafterabbreviated to CAS) is a 2-oxoglutarate linked oxygenase which convertsproclavaminic acid into clavaminic acid (these are intermediates in theclavulanic acid biosynthetic pathway). For further details see papers byS. W. Elson et al in J. Chem. Soc. Chem. Commun., 1987, pages 1736,1738, and 1739. See also European Patent Application Publication No. 0213 914.

In order to clearly define the invention reference is made to theaccompanying drawings in which:

FIG. 1 is a restriction map of DNA I

FIG. 2 is a restriction map of DNA II

FIG. 3 is a restriction map of DNA III

FIG. 4 is a restriction map of seven restriction fragments derived fromDNA III

FIG. 5a is a restriction map of the recombinant plasmid pBROC 41A;

FIG. 5b is a restriction map of the recombinant plasmid pBROC 41B; and

FIG. 6 is a restriction map of the recombinant plasmid pBROC 31.

In the Figures the abbreviations Eco RI, Pst I etc. are conventionalabbreviations for restriction endonucleases, and the approximate lengthin kilobases (Kb) of the DNA, as determined by sizing experimentscarried out by agarose gel electrophoresis, is indicated. It should beunderstood that the Figures are not necessarily intended to show all thepossible restriction sites present on the DNA fragments illustrated.

In a first aspect, the present invention provides DNA comprising asequence which encodes at least one enzyme involved in clavulanic acidbiosynthesis, characterised in that the DNA is approximately 60kb inlength and has the configuration of restriction sites shown in DNAfragment (I) in FIG. 1.

It will be appreciated that the DNA (I) of the invention is not in its`natural` state (i.e. as found in the chromosomal DNA of S.clavuligerus) but has been purified and isolated to separate it fromflanking DNA.

The advantage of the invention is that DNA (I) comprises one or moregenes involved in clavulanic acid biosynthesis which can be utilised, ashereinbelow described, to increase the yield of clavulanic acid producedby a clavulanic acid producing organism. A particular advantage is thatthe titre of clavulanic acid can be substantially increased in wild-typeStreptomyces species, for example S. clavuligerus ATCC 27064, whichnaturally tend to produce only fairly low levels of clavulanic acid.

Certain restriction fragments of the DNA(I) may also be utilised in theabove manner provided they contain one or more intact genes involved inclavulanic acid biosynthesis.

Accordingly, the present invention further provides DNA which is asubfragment of the DNA (I) shown in FIG. 1, with the proviso that if thesubfragment is a subfragment of the DNA (II) shown in FIG. 2, thesubfragment is identical with or is a subfragment of the DNA (III) shownin FIG. 3.

The subfragment according to the invention may be derived by cleavingthe DNA (I) with appropriate restriction enzymes by known methods.Subfragments according to the invention may also be prepared by cleavinglarger DNA subfragments with appropriate restriction enzymes or byligating smaller subfragments using known methods.

A preferred subfragment has the configuration of restriction sites shownin DNA (III) in FIG. 3 or is a subfragment thereof.

Particular subfragments of the DNA (III) include those shown in FIG. 4,viz:

    ______________________________________                                        a)     EcoRI - BamHI fragment (IV);                                           b)     BclI - BclI   fragment (V);                                            c)     BclI - BclI   fragment (VI);                                           d)     BglII - BglII fragment (VII);                                          e)     BglII - BglII fragment (VIII);                                         f)     BglII - BglII fragment (IX);                                           g)     SphI - Sph I  fragment (X).                                            ______________________________________                                    

By means of suitable experiments described hereinbelow it has been foundthat the DNA (I) shown in FIG. 1 comprises a sequence encoding CASactivity.

Accordingly, in another aspect, the present invention provides DNAcomprising a gene encoding a protein with clavaminic acid synthaseactivity.

The gene for CAS activity is located within the DNA fragment (IX) shownin FIG. 4, and accordingly, DNA fragment (IX) is another preferredsubfragment according to the invention.

It will be understood that the invention encompasses DNA which may nothave the precise configuration of restriction sites illustrated in FIGS.1 to 4 if the said DNA has been derived by standard techniques includingnucleotide deletion, substitution, addition or inversion fron the DNAaccording to any aspect of the invention described above.

Preferably the DNA of this invention is derived from S. clavuligerusATCC 27064 and FIGS. 1 to 4 show S. clavuligerus DNA.

However the invention also encompasses DNA sequences obtained fromsuitable clavulanic acid producing organisms other than S. clavuligerus,which sequences do not have the configuration of restriction sitesillustrated in FIGS. 1-4 but which hybridise, preferably underconditions of high stringency, with the DNA (III) shown in FIG. 3, or asubfragment thereof, and which code for an enzyme involved in clavulanicacid biosynthesis.

Other known clavulanic acid producing organisms include S. jumonjinensisATCC 29864 and S. katsurahamanus T-272.

In a further aspect the invention provides recombinant DNA comprisingthe DNA of any aspect of the invention as hereinabove described.

Preferably the recombinant DNA of the invention comprises a recombinantvector, more preferably a vector capable of transforming and undergoingautonomous replication in a clavulanic-acid producing organism or avector from which insert DNA can be integrated into the chromosome ofthe clavulanic acid producing organism via homologous recombination.

In one preferred aspect the recombinant vector is a high expressionvector.

The DNA according to any aspect of this invention may be introduced intoany suitable vector by methods well known in the art, for example bydirect combination of cohesive ends, homopolymer tailing, or by means ofa linker or adaptor molecule.

In one preferred aspect, the vector is derived from a Streptomycete.

Specific examples of such vectors include the following

(1) pIJ 913 (molecular weight 15.7 megadaltons), a low copy numbervector described in Lydiate, D. J., et al., Gene (1985), 35, 223-235;

(2) pIJ 702 (molecular weight 3.7 megadaltons), a high copy numbervector described in Katz, E. et al, J. Gen. Microbiol (1983), 129,2703-2714.

(3) pIJ 680 described by Hopwood et al (1985) Genetic Manipulation ofStreptomyces. A Laboratory Manual. The John Innes Foundation.

The vector may also be derived from a non-Streptomycete; for example thecosmid pTCF (Grosveld et al., Nucleic Acids Research, 1982, 10,6715-6732) or the plasmid pAT 153 (Twigg, A. J. and Sherratt, D.,Nature, 1980, 283, 216-218).

It will be appreciated that recombinant vectors prepared according tothe above methods may contain the insert DNA in one of two possibleorientations. Recombinant vectors containing both orientations areincluded within the scope of the invention. Thus, for example, when theinsert DNA fragment has the configuration of restriction sites shown in(IV) (FIG. 4) and the plasmid vector into which it is introduced is pIJ913, the restriction maps of the resulting recombinant plasmids,referred to as pBROC 41A and pBROC 41B, are shown in FIGS. 5(a) and5(b). Of these plasmids, pBROC 41B FIG. 5(b)! is preferred.

A preferred recombinant vector which may be prepared by inserting theDNA (III) in FIG. 3 into pAT 153 is shown in FIG. 6 and is designatedpBROC 31.

Other preferred recombinant vectors according to the invention includethose designated pWOR10 (constructed by ligating DNA fragment IX ashereinabove defined into the Bam HI site of pIJ680) and pBROC44(constructed by ligating the same DNA fragment IX into the Bql II siteof pIJ702). In pWOR10 the unique Bcl I site in fragment IX is 2.45Kbfrom the unique Xho I site in the pIJ 680 vector. In pBROC44 the uniqueBcl I site in fragment IX is 3Kb from the unique Bam HI site of the pIJ702 vector.

The insert DNA in one vector may be sub-cloned into another vector bystandard procedures. For example, the recombinant plasmid pBROC 41Bcharacterized as shown in FIG. 5(b), may be obtained by:

a) isolating the large Eco RI--Eco RI segment (14.35Kb) from pBROC 31(FIG. 6) by cleavage with Eco RI; and

b) ligating the above Eco RI--Eco RI segment to pIJ 913, linearized bycleavage with Eco RI.

To prepare the DNA and recombinant vectors of the invention, a randomarray of chromosomal DNA fragments may be generated by partial digestionof S. clavuligerus (ATCC 27064) DNA by any convenient restrictionenzyme. The endonuclease Mbo I and its isoschizomers may be particularlysuitable for this purpose.

The DNA fragments may then be size-fractionated on a salt gradient andfractions containing fragments around 35-45Kb in length may be taken(see Grosveld et al, Nucleic Acids Research, 1982, 10, 6715-6732). TheDNA fragments may then be ligated by conventional `shot-gun` methods toa cleaved vector to form a `clone bank`.

If desired, smaller fragments, for example of length 10 Kb or more, maybe obtained by size fractionation on an agarose gel usingelectrophoretic elution. These smaller DNA fragments may then be used toform a clone bank as described above.

The vector used to form the clone bank may be a cosmid (able to carrylarge pieces of DNA), and for this purpose the cosmid pTCF (see Grosveldet al, loc. cit.) is particularly suitable.

The plasmid pAT 153 may also advantageously be used in forming a clonebank to carry smaller pieces of DNA.

A clone bank prepared as described above may then be `probed` byconventional methods using as a hybridization probe a piece of DNAcapable, when inserted into a suitable vector and transformed into anon-producer mutant strain of a clavulanic acid-producing host (a`blocked` mutant) of restoring the ability of the host to synthesizeclavulanic acid. It will be appreciated that the probe will beradiolabelled, for example with ³² P, and may either comprise the saidpiece of DNA itself or consist of a vector containing the said DNAprovided that the said vector has no appreciable homology with thevector used to form the clone bank to be probed.

Suitable probes may be isolated by the procedure described by C. R.Bailey et al. in Biotechnology, 1984, 2, 808-811.

A preferred probe is the plasmid pWOR1 described by C. R. Bailey et al.(loc. cit.).

To carry out the procedure described in Biotechnology, 1984, 2, 808-811,it is necessary to have access to S. clavuligerus dc1C8, a mutant strainof S. clavuligerus which lacks the ability to produce clavulanic acid.The details relating to the deposition, derivation and characteristicsof S. clavuligerus dc1C8 are as follows.

Deposition

The strain S. clavuligerus dc1C8 was deposited in the NationalCollection of Industrial and Marine Bacteria, Aberdeen, Scotland on 19thFebruary, 1986, the deposit (accession number NCIB 12209) being madeunder the terms of the Budapest Treaty on the International Recognitionof the Deposit of Micro-organisms for the Purposes of Patent Procedure.

Derivation of S. clavuligerus NCIB 12209

Breaking out a sample of S. clavuligerus ATCC 27064 showed a variety ofmorphologies. Fourteen different types were recognised. One of these wasisolated as S. clavuligerus SC2.

Treatment of S. clavuligerus SC-2 with 10 μM of ethidium bromide wasthen carried out on petri dishes and the plates were incubated for 10days. Survivors were assayed for clavulanic acid production. One isolatewhich produced no detectable clavulanic acid when assayed by HPLC(detection at 230 mm) was designated as strain dc1C8 and deposited underaccession number NCIB 12209 as hereinabove described. S. clavuligerusNCIB 12209 forms a further aspect of the present invention.

Taxonomy of S. clavuligerus NCIB 12209

The taxonomical and morphological properties of S. clavuligerus NCIB12209 were found to be essentially similar to those of S. clavuligerusATCC 27064, a description of which may be found in U.S. Pat. No.3,862,008 and also in Higgens, C. E. and Kastner, R. E., Int. J.Systematic Bacteriol., 1971, 21, 326-331.

After probing a clone bank prepared as described above with a suitablehybridization probe, for example pWOR1, insert DNA corresponding to, orembracing, the DNA of the invention may then be sub-cloned by standardmethods, or cleaved as hereinbefore described.

In the event that an inappropriate or too short piece of DNA is obtainedfrom the probing step, the newly isolated DNA fragment may itself beused as a hybridization probe (after radio-labelling by conventionalmethods, eg. nick-translation) to identify further positive colonies inthe clone bank, the insert DNA of which may be analysed by restrictionmapping and if necessary, modified by cleavage with appropriaterestriction enzymes.

The above process may be repeated until the required DNA according tothe invention is isolated.

In yet a further aspect the invention provides a host transformed by arecombinant vector of the invention.

The host may be transformed by standard techniques.

In one aspect the host may be a non-producer of clavulanic acid, forexample E. coli or S. lividans. Such hosts may be of value in geneticmanipulation procedures and can be advantageously used to express largequantities of enzymes involved in clavulanic acid biosynthesis, forexample an enzyme having CAS activity.

Preferred hosts, however, are those which are natural producers ofclavulanic acid when cultured in an appropriate medium, or non-producingmutants thereof blocked in clavulanic acid biosynthesis which the DNAaccording to the invention can then repair so that clavulanic acidsynthesis is restored and preferably enhanced. Preferred hosts of thistype are Streptomycetes including S. clavuligerus ATCC 27064, S.jumonjinensis ATCC 29864 and S. katsurahamanus T-272 or are derivedtherefrom.

The advantage of employing such hosts is that under appropriateconditions an increased titre of clavulanic acid can be obtained aftertransformation with a recombinant vector according to the invention.

Accordingly the present invention further provides a method forproducing clavulanic acid in a host which is naturally a producer ofclavulanic acid or from a blocked non-producing mutant of a said host,which method comprises the steps of:

(a) transforming the said host or said non-producing mutant thereof witha recombinant vector according to the invention; and

(b) culturing the transformants so formed under appropriate conditionsso that production of clavulanic acid takes place.

General methods for culturing a clavulanic acid-producing organism so asto obtain clavulanic acid are given in U.K. Patent Specification No.1,508,977.

In the above method the host is preferably S. clavuligerus ATCC 27064,or is derived therefrom.

Preferably the recombinant vector used in the above method is pWOR10 ashereinabove defined.

The following examples illustrate the invention.

EXAMPLE 1 Construction of a library of S. clavuligerus ATCC 27064 DNA inthe cosmid vector pTCF

S. clavuligerus ATCC 27064 chromosomal DNA was isolated as described inEuropean Patent Application Publication No. 0 233 715. Three aliquots of20 μg of chromosomal DNA were partially digested with Mbo I (0.5 unitsper μg of DNA) for 5 minutes, 10 minutes and 15 minutes under standardconditions. The three aliquots were pooled and fractionated on a salt(1.25M-5M NaCl)/Tris EDTA gradient and centrifuged for 3 hours at 39Krpm in a SW40.1 Beckman rotor. Twelve fractions were collected and theisolated DNAs from fractions 5-9 yielded 10 μg Mbo I DNA fragmentsaround 35-45kb in size.

The preparation of pTCF vector arms was carried out according toGrosveld, F. G., Lund, T., Murray, E. J., Mellor, A. L., Dahl, H. H.,Flavell, R. A., Nucleic Acids Research (1982), 10, 6715-6732.

S. clavuligerus DNA fragments and vector arms were ligated in a ratio of5:3:3, the mix packaged and used in the transduction of E.coli ED8767 asdescribed in Grosveld et al. (loc. cit.). This procedure gave 4×10⁶colonies per μg packaged DNA.

EXAMPLE 2 Preparation of DNA I

5000 E. coli ED8767 colonies containing pTCF with S. clavuligerus ATCC27064 DNA inserts were immobilised and lysed on nitrocellulose filters.The plasmid pWOR1 (Bailey, C. R. et al. (loc. cit.) was isolated, nicktranslated and used to probe the filters by standard colonyhybridisation techniques.

Two hybridising colonies were obtained which, when combined, contain the60 kb DNA I segment illustrated in FIG. 1.

EXAMPLE 3 Construction of a library of S. clavuligerus ATCC 27064 DNA inpAT153

S. clavuligerus ATCC 27064 chromosomal DNA was isolated as described inEuropean Patent Application Publication 0 233 715.

60 μg of S. clavuligerus ATCC 27064 DNA was partially digested with MboI and fractionated on an agarose gel. DNA was isolated from fractionscontaining fragments of >10 kb in size by electrophoretic elution asdescribed by Maniatis, T., Fritsch, E. F., Sambrook, J. (1982) MolecularCloning; A Laboratory Manual (Cold Spring harbor Laboratory).

10 μg of pAT153 (Twigg and Sherratt (loc. cit.)) was digested with BamHI and treated with calf intestinal alkaline phosphatase to removeterminal phosphate groups and prevent recircularisation. The vectorand >10 kb fragments of S. clavuligerus ATCC 27064 DNA were ligatedtogether and used to transform E. coli DH1.

EXAMPLE 4 Preparation of DNA III

6000 E. coli DH1 colonies containing pAT153 with S. clavuligerus ATCC27064 inserts were immobilised and lysed on nitrocellulose filters. The2.2 kb Sph I--Bgl II insert fragment from pWOR1 sites 3-4 in FIG. 3 ofBailey et al (loc. cit.)! was isolated, nick translated and used toprobe the filters by standard colony hybridisation techniques.

One hybridising colony was obtained which contains the 13.1 kb DNA IIIfragment illustrated in FIG. 3.

EXAMPLE 5 Enhancement of clavulanic acid titre in S. clavuligerus strainSC2 by the plasmid PWOR10

Plasmid pWOR10 was constructed by ligating the Bql II-Bql II fragment IX(FIG. 4) into the Bam H1 site of pIJ680 (Hopwood et al., 1985 GeneticManipulation of Streptomyces. A Laboratory Manual. The John InnesFoundation).

Plasmid pWOR10 was transformed into S. clavuligerus SC2 (reisolate ofwild type) by a method analogous to that described in C. R. Bailey et al(loc cit.). Transformants were picked off and restreaked on M5D medium(Dextrin, 1.0%; K₂ HP04, 0.1%; MgSO4, 0.1%; NaCl, 0.1%; (NH4)₂ SO4,0.1%; CaC03, 0.4%; Trace Elements (FeSO4 0.0001%; MnCl₂, 0.0001%; ZnSO4,0.0001% ,; Agar, 2.0%) containing 5 μg/ml thiostrepton. Cells of eachtransformant were stabbed onto M5D and grown for 6 days at 26° C.

The bioassay plates were then overpoured with soft blood agar (Oxoid)containing Klebsiella aerogenes strain as described in Reading, C. andCole, M. (1977) Antimicrob. Agents Chemother, 11, 852-857, 4%tetrazolium salts and 5 μg/ml penicillin G. After overnight incubationat 26° C. zones of inhibition were measured. Those transformants showingzones greater than the control culture were transferred to shake-flaskculture for accurate titre assessment. Cells are inoculated into 25 mlof a suitable seed medium as described in U.K. Patent Specification No.1,508,977 and grown for 3 days at 26° C. with shaking. 1 ml of seedculture is inoculated into a final stage medium as described in U.K.Patent Specification No. 1,508,977 and grown at 26° C. for four days.

Samples of culture broth were withdrawn after three and four days growthand assayed for clavulanic acid productivity as described in Bird, A. E.et al (1982) Analyst, 107, 1241-1245 and Foulston M., and Reading, C.(1982) Antimicrob Agents Chemother., 22, 753-762.

Results

Of the 21 isolates tested, one gave enhanced titres compared to S.clavuligerus SC2 yielding up to 53% advantage in cultures grown in theabsence of thiostrepton and 39% advantage in cultures grown in thepresence of thiostrepton. In all cases autonomous pWOR10 was found to bepresent.

EXAMPLE 6 Transformation of S. lividans with pBROC44

pBROC 44 was prepared by ligating the Bql II--Bql II fragment IX (FIG.4) in to the Bql II site of pIJ 702 (see Hopwood et al. loc. cit.)

S. lividans spores were inoculated into 25 ml of protoplast seed medium(tryptone soya broth +1% maltose) in a 250 ml shake flask and incubatedfor 3 days at 30° C. with shaking. 1 ml of seed culture was transferredto 25 ml final stage protoplasting medium comprising 15 ml YEME (Hopwoodet al., loc. cit) with 1% maltose plus 10 ml TSB plus 0.5% glycine plus1% maltose! in 250 ml shake flask containing a coiled spring. Incubationwas overnight (16 h) at 30° C. The culture was harvested in sterilecentrifuge tubes and mycelium washed in 10.3% sucrose. The cell pelletwas resuspended in 1.5 ml lysozyme mix and incubated at 25° C. for 1hour, mixing occasionally. Protoplast formation was monitored using amicroscope. Protoplast buffer (PB) (4 ml) (Hopwood et al., loc. cit.)was added and mixed and the cell suspension was then filtered throughsterile cotton wool filters. The protoplast suspension was centrifuged,washed briefly in PB and finally resuspended in 2 ml PB. Protoplastconcentration was measured using a Thoma counting chamber.

4×10⁹ protoplasts were placed in fresh centrifuge tube, 5 ml of PB addedand pelleted by centrifugation. Protoplasts were resuspended in approx.400 μl of PB. 5 μl of ligation mix was added to the protoplastsuspension and within 30 seconds 0.5 ml transformation buffer (Hopwoodet al. loc. cit., page 246) was added. After 10 seconds 5 ml of PB wasadded and the protoplasts were centrifuged.

Protoplasts were resuspended in 1 ml PB and dilutions made in PB down to10⁻³. 0.1 ml samples of each dilution were plated out onto regenerationmedium R2YE; see Thompson, C. J., Ward, J. M. and Hopwood, D. A (1980)Nature, 286, 525-527. Agar plates were incubated at 30° C. After 20hours plates were overpoured with 2 ml soft nutrient agar containing 500μg/ml thiostrepton. Agar plates were incubated for a further 3 days.Thiostrepton resistant transformants were picked off regeneration platesand restreaked on fresh R2YE agar. After 3-4 days growth transformantswere inoculated into seed medium containing 50 μg/ml thiostrepton andgrown with shaking for 2 days at 30° C. Plasmid was isolated accordingto the method of Hopwood et al (loc. cit). The correct constructdesignated pBROC44 was confirmed by restriction endonuclease digestionas described by Maniatis et al (loc. cit) (1982).

EXAMPLE 7 The demonstration of clavaminic acid synthase activity inpBROC44-transformed S. lividans

The experiments described below demonstrate the presence of clavaminicacid synthase (CAS) activity in S. lividans that had been transformed bypBROC44 (Example 6). Three cultures were examined:

(i) S. lividans host;

(ii) S. lividans containing pIJ702, and

(iii) S. lividans containing pBROC44 (Example 6).

Cultures (i) and (ii) acted as negative controls for (iii).

Because the vector pIJ702 contains a locus for thiostrepton resistance,the two cultures containing this vector were grown in the presence ofthiostrepton (50 μg/ml). The host itself was grown without thiostrepton.

The medium used was as follows:

    ______________________________________                                        Difco yeast extract                                                                              3 g)                                                       Difco bacto peptone                                                                              3 g)                                                       Oxoid malt extract 3 g)    1 liter H.sub.2 O                                  Glucose            10 g)                                                      Sucrose            340 g)                                                     ______________________________________                                    

and was supplemented after autoclaving with MgCl₂ and glycine to finalconcentrations of 5 mM and 50 mM, respectively.

The medium was inoculated with a single agar plug from a spore plate,and cultures were grown in 250 ml or 500 ml sprung shake-flasks at 31°C.

The mycelium was harvested by centrifugation after 48 hr. growth, andwas sonicated (3×5 sec. bursts) to release intracellular enzymes in thesame volume of 50 mM TRIS HCl/10 mM MgCl₂ /10 mM KCl/10% glycerol/1 mMphenylmethylsulphonyl fluoride (PMSF) /pH 7.5.

To assay for CAS activity, the centrifuged sonicate (115 μl) wasincubated with Fe²⁺ (15 μl, 10 mM), α-ketoglutarate (15 μl, 10 mM) andthe substrate for CAS 5-amino-3-hydroxy-2-(2-oxoazetidin-1-yl)valerate;proclavaminic acid; as described in EP-A-O 213 914! (5 μl, 5 mg/ml) for5 minutes at room temperature, followed by incubation with imidazole(37.5 μl, 20%, pH 6.8) for 10 minutes, then detection of the derivatisedproduct by HPLC using a 30 cm Waters Cl8 column equilibriated in 0.1MNaH₂ PO₄ /6% MeOH/pH 3.2 at 2 mls/min., detecting at 312 nm. Soft gelpermeation chromatography was performed at 4° C. using a 2.6×45 cmcolumn of Ultrogel AcA 54 equilibrated in sonication buffer (see above)at 20 mls/hr. Fractions were assayed for CAS activity as above. Proteinassays were performed according to the method of Bradford (1976)Analytical Biochem. 72, 248-254.

Results

Cultures of S. lividans alone, S. lividans containing the vector pIJ702,and S. lividans transformed with pBROC44 were grown for 48 hours to givean opaque broth. The mycelium of each culture was harvested andsonicated, and the centrifuged sonicates were assayed for CAS activity.HPLC data showed that the S. lividans transformed with pBROC44 gave riseto an HPLC peak with the following characteristics:

(i) it co-elutes (2.5 mins) with the standard for clavaminic acid.

(ii) it is absent from the two control cultures (S. lividans alone andS. lividans containing pIJ702 without the insert);

(iii) it is absent when the pBROC44-transformed culture is assayed inthe absence of either Fe²⁺, α-ketoglutarate or the substrate.

This evidence shows that the pBROC44-transformed culture gives rise toCAS activity.

The specific activity of the CAS purified from S. lividans/pBROC44 wasfound to be 0.0036 μmole/min/mg protein. This compares with valuesbetween 0.008 and 0.06 μmole/min/mg protein for CAS from S. clavuligerusATCC 27064 (reisolate SC2). Although the specific activity of the clonedCAS is slightly less than that of native CAS, the data indicate that theenzyme is translated intact, and that pBROC44 contains the complete CASgene.

We claim:
 1. A purified and isolated DNA comprising a DNA sequencederivable from S. clavuligerus, which DNA encodes at least one enzymeinvolved in clavulanic acid biosynthesis, characterized in that said DNAis approximately 60 kb in length and has the configuration ofrestriction sites shown in DNA fragment (I) in FIG.
 1. 2. The purifiedand isolated DNA according to claim 1 comprising a gene encoding aprotein with clavaminic acid synthase activity.
 3. The purified andisolated DNA sequence according to claim 2 which hybridizes to the DNAfragment (III) having the configuration of restriction sites shown inFIG. 3, or a DNA subfragment selected from the group consisting ofsubfragments (IV), (V), (VI), (VII), (VIII), (IX), and (X) shown in FIG.4 and which DNA sequence codes for an enzyme having clavaminic acidsynthase activity.
 4. An isolated and purified DNA encoding at least oneenzyme involved in clavulanic acid biosynthesis, which DNA has beenderived by way of nucleotide deletion, substitution, insertion, orinversion from the DNA claimed in claim
 1. 5. The purified and isolatedDNA according to claim 4 which is derived from S. clavuligerus.
 6. Arecombinant DNA comprising DNA as claimed in claim
 5. 7. A recombinantvector comprising the recombinant DNA of claim
 6. 8. The recombinantvector according to claim 7 which is designated pWOR10, pBROC44,pBROC31, pBROC41A or pBROC41B.
 9. The recombinant vector according toclaim 7 which is a high expression vector.
 10. A host transformed with avector as claimed in claim
 7. 11. The transformed host as claimed inclaim 10 which is a Streptomycete or E.coli.
 12. The Streptomycete hostas claimed in claim 11 which is naturally a producer of clavulanic acid.13. A subfragment of the DNA of claim 1 encoding at least one enzymeinvolved in clavulanic acid biosynthesis, which subfragment is the DNAfragment (III) shown in FIG. 3 or is a subfragment thereof selected fromthe group consisting of subfragments (IV), (V), (VI), (VII), (VIII),(IX), and (X) shown in FIG.
 4. 14. The DNA subfragment as claimed inclaim 13 in which the DNA has the configuration of restriction sitesshown in DNA fragment (IX) in FIG.
 4. 15. The DNA subfragment accordingto claim 13 which has been purified and isolated, comprising a geneencoding a protein with clavaminic acid synthase activity.
 16. Anisolated and purified DNA encoding at least one enzyme involved inclavulanic acid biosynthesis, which DNA has been derived by way ofnucleotide deletion, substitution, insertion, or inversion from the DNAas claimed in claim
 13. 17. A recombinant DNA comprising the DNA claimedin claim
 16. 18. A recombinant vector comprising the recombinant DNA ofclaim
 17. 19. A vector according to claim 18 which is a high expressionvector.
 20. A host transformed with the vector of claim
 18. 21. A hosttransformed with the vector of claim
 19. 22. An isolated and purifiedDNA encoding at least one enzyme involved in clavulanic acidbiosynthesis, which DNA has been derived by way of nucleotide deletion,substitution, insertion, or inversion from the DNA as claimed in claim2.
 23. A recombinant DNA comprising the DNA claimed in claim
 22. 24. Arecombinant vector comprising the recombinant DNA of claim
 23. 25. Avector according to claim 24 which is a high expression vector.
 26. Ahost transformed with the vector of claim
 24. 27. A host transformedwith the vector of claim
 25. 28. An isolated and purified DNA encodingat least one enzyme involved in clavulanic acid biosynthesis, which DNAhas been derived by way of nucleotide deletion, substitution, insertion,or inversion from the DNA as claimed in claim
 14. 29. A recombinant DNAcomprising the DNA claimed in claim
 28. 30. A recombinant vectorcomprising the recombinant DNA of claim
 29. 31. A vector according toclaim 30 which is a high expression vector.
 32. A host transformed withthe vector of claim
 30. 33. A host transformed with the vector of claim31.
 34. An isolated and purified DNA encoding at least one enzymeinvolved in clavulanic acid biosynthesis, which DNA has been derived byway of nucleotide deletion, substitution, insertion, or inversion fromthe DNA as claimed in claim
 3. 35. A recombinant DNA comprising the DNAclaimed in claim
 34. 36. A recombinant vector comprising the recombinantDNA of claim
 35. 37. A vector according to claim 36 which is a highexpression vector.
 38. A host transformed with the vector of claim 36.39. A host transformed with the vector of claim
 36. 40. The purified andisolated DNA according to claim 1 which is derived from S. clavuligerus.41. The DNA subfragment according to claim 13 which is derived from S.clavuligerus.
 42. The purified and isolated DNA according to claim 2which is derived from S. clavuligerus.
 43. The purified and isolated DNAas claimed in claim 2 in which the DNA has the configuration ofrestriction sites shown in DNA fragment (IX) in FIG.
 4. 44. The DNAsubfragment according to claim 6 which is derived from S. clavuligerus.45. The purified and isolated DNA according to claim 33 which is derivedfrom S. clavuligerus.
 46. A method for producing clavulanic acid in ahost which is naturally a producer of clavulanic acid or from a blockednon-producing mutant of a said host, which method comprises the stepsof:(a) transforming the said host or said non-producing mutant thereofwith a vector as claimed in any one of claims 7 to 9; and (b) culturingthe transformants so formed under appropriate conditions so thatproduction of clavulanic acid takes place.
 47. The method as claimed inclaim 46 in which the transformants comprise a host selected from thegroup consisting of S. clavuligerus ATCC 27064, S. jumonjinensis ATCC29864, S. katsurahamanus T-272, or a mutant thereof.